1. Field of the Invention
This invention relates to the production of polyhydroxyalkanoate by the culture of microorganisms which produce same.
Poly-3-hydroxybutyrate is a linear polyester of D(xe2x88x92)-3-hydroxybutyrate. It was first discovered in Bacillus megaterium in 1925. Polyhydroxy-butyrate accumulates in intracellular granules of a wide variety of bacteria. The granules appear to be membrane bound and can be stained with Sudan Black dye. The polymer is produced under conditions of nutrient limitation and acts as a reserve of carbon and energy. The molecular weight of the polyhydroxybutyrate varies from around 50,000 to greater than 1,000,000, depending on the microorganisms involved, the conditions of growth, and the method employed for extraction of the polyhydroxybutyrate. Polyhydroxybutyrate is an ideal carbon reserve as it exists in the cell in a highly reduced state, (it is virtually insoluble), and exerts negligible osmotic pressure.
Polyhydroxybutyrate and related poly-hydroxy-alkanoates, such as poly-3-hydroxyvalerate and poly-3-hydroxyoctanoate, are biodegradable thermo-plastics of considerable commercial importance.
The term xe2x80x9cpolyhydroxyalkanoatexe2x80x9d as used hereinafter includes copolymers of polyhydroxy-butyrate with other polyhydroxyalkanoates such as poly-3-hydroxyvalerate.
2. Background Information
Polyhydroxyalkanoate is biodegradable and is broken down rapidly by soil microorganisms. It is thermoplastic (it melts at 180xc2x0 C.) and can readily be moulded into diverse forms using technology well-established for the other thermoplastics materials such as high-density polyethylene which melts at around the same temperature (190xc2x0 C.). The material is ideal for the production of biodegradable packaging which will degrade in landfill sites and sewage farms. The polymer is biocompatible, as well as biodegradable, and is well tolerated by the mammalian, including human, body, its degradation product, 3-hydroxybutyrate, is a normal mammalian metabolite. However, polyhydroxyalkanoate degrades only slowly in the body and its medical uses are limited to those applications where long term degradation is required.
Polyhydroxyalkanoate, produced by the microorganism Alcaligenes eutrophus, is manufactured, as a copolymer with of polyhydroxy-butyrate and polhydroxyvalerate, by Imperial Chemical Industries PLC and sold under the Trade Mark BIOPOL. It is normally supplied in the form of pellets for thermoprocessing. However, polyhydroxyalkanoate is more expensive to manufacture by existing methods than, say, polyethylene. It is, therefore, desirable that new, more economic production of polyhydroxy-alkanoate be provided.
An object of the present invention is to provide materials and a method for the efficient production of polyhydroxyalkanoate.
According to the present invention there are provided gene fragments isolated from the bacterium Chromatium vinosum and encoding PHA polymerase, acetoacetyl CoA reductase and xcex2-ketothiolase.
Preferably the C. vinosum is of the strain designated D, available to the public from the Deutsche Sammlung fur Mikroorganismen under the Accession Number 180.
The invention also provides a 16.5 kb EcoR1 fragment of C. vinosum DNA, designated PP10, hybridizable to a 5.2 kb SmaI/EcoR1 fragment, designated SE52 isolated from Alcaligenes eutrophus and known to contain all three of said genes responsible for expression of PHAS.
The invention further provides a fragment of the said PP10 fragment, designated SE45, encoding the PHA-synthase and xcex2-ketothiolase genes and a region, designated SB24, encoding the acetoacetyl CoA reductase gene.
The invention also provides a recombinant genome of a microorganism, preferably a bacterium or a plant, which contains one or more of said fragments designated PP10, SE45 and region SB24.
Finally, the invention provides a method for the manufacture of PHAs, comprising culturing the microorganism Chromatium vinosum, or a bacterium of a different species having stably incorporated within its genome by transformation one or more PHA synthesising genes from Chromatium vinosum. 
The biosynthesis of polyhydroxyalkanoate from the substrate, acetyl-CoA involves three enzyme-catalysed steps.
The three enzymes involved are xcex2-ketot hiolase, acetoactyl-CoA-reductase and polyhydroxy-butyrate-synthase, the genes for which have been cloned from Chromatium vinosum. The three genes are known to facilitate production of polyhydroxyalkanoates, the reactions involved being represented as follows: 